Abstract
Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3′-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI.
Highlights
The involvement of miRNAs in tooth development was first proposed in 200817
The morphologies of mouse enamel organs overlying mandibular first molars and incisors were first revisited by staining the PN6 and PN9 mandible section slides with U6 LNA-DIG probes (Fig. 1a–e), which indicated a positive staining pattern of nuclei
Enamel formation is a mineralization process consisting of secretory and maturation stages
Summary
The involvement of miRNAs in tooth development was first proposed in 200817. Two functional studies, in which epithelial Dicer-1 was deleted at earlier stages of tooth development, elicited tooth phenotypes with varying severities[18,19]. We identified a group of miRNAs expressed in a stage-specific manner, which are significantly enriched in the gene functional categories equivalent to those key processes during amelogenesis. MiR-153 is predicted to be one of the miRNA regulators targeting endocytotic and endosomal/lysosomal pathways, and experimental evidence from luciferase report assays validated the predicted interaction between miR-153 and the 3′-UTR of Lamp[111]. We further extended our knowledge about the role of miR-153 in amelogenesis and the pathogenesis of Amelogenesis Imperfecta (AI) by collecting additional evidence from both in vitro and in vivo functional studies. We identified key genes in the endocytotic and endosomal/lysosomal pathways that were regulated by miR-153 expression in our assay systems. We provide evidence of the importance of miR-153 expression levels in regulating amelogenesis and the specific molecular mechanisms that miR-153 plays in this process. Our findings would help better understand the potential etiologic factors contributing to the occurrence of AI and possible preventative or therapeutic approaches to disease
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